Ruthenium (ii) compounds for use in the therepy of cancer

ABSTRACT

Compounds which may be used in the treatment and/or prevention of cancer have the formula (I): wherein R 1  and R 2  together with the ring to which they are bound represent a saturated or unsaturated carbocyclic or heterocyclic group.

[0001] This invention relates to ruthenium (II) compounds, to their use in medicine, particularly for the treatment and/or prevention of cancer, and to a process for their preparation.

[0002] Certain ruthenium (II) complexes have been proposed for use in treating cancer. For example, U.S. Pat. No. 4,980,473 discloses 1, 10-phenanthroline complexes of rutheniunm (II) and cobalt (III) which are said to be useful for the treatment of tumour cells in a subject.

[0003] Some other ruthenium (II) and ruthenium (III) complexes which have been shown to exhibit antitumour activity are mentioned in Guo et al, Inorganica Chimica Acta, 273 (1998), 1-7, specifically trans-[RuCl₂(DMSO)₄], trans-[RuCl₂(imidazole)₂]- and trans-[RuCl₄(indazole)₂]Guo et at discloses that the most interesting feature of these complexes is their anti-metastatic activity. Clarke et al have reviewed the anticancer, and in particular the antimetastatic, activity of ruthenium complexes: Chem. Rev. 1999, 99, 2511-2533. Also, Sava has reviewed the antimetastatic activity in “Metal Compounds in Cancer Therapy” Ed by S P Fricker, Chapman and Hall; London 1994, p. 65-91.

[0004] Dale. et al, Anti-Cancer Drug Design (1992), 7, 3-14, describes a metronidazole complex of ruthenium (II) ie, [(η⁶-C6H₆)RuCl₂(metronidazole)] and its effect on DNA and on E. coli growth rates. Metronidazole sensitises hypoxic tumour cells to radiation and appears to be an essential element of the complexes of Dale et al. There is no indication in Dale et al that the complexes would be at all effective in the absence of the metronidazole ligand.

[0005] Kramer et al, Chem Eur J., 1996, 2, No. 12, p. 1518-1526 discloses half sandwich complexes of ruthenium with amino esters.

[0006] There exists a need for novel and-cancer compounds which can be used as alternatives to the compounds which are currently available.

[0007] The present invention provides a novel class of ruthenium (II) complexes having anti-tumour activity.

[0008] According to the present invention there is provided a ruthenium (II) compound of formula (I):

[0009] wherein R¹ and R² together with the ring to which they are bound represent a saturated or unsaturated carbocyclic or heterocyclic group containing up to three 3- to 8- membered carbocyclic or heterocyclic rings, wherein each carbocyclic or heterocyclic ring may be fused to one or more other carbocyclic or heterocyclic rings; and wherein each of the rings may be optionally substituted by one or more groups independently selected from alkyl, aryl, alkaryl, halo, carboxy, carboxyester, carboxyamide, sulfonate, sulfonido or alkether;

[0010] R³, R⁴, R⁵ and R⁶ independently represent H, alky, —CO₂R′, aryl or alkaryl, which latter two groups are optionally substituted on the aromatic ring;

[0011] R′ represents alkyl, aryl or alkaryl;

[0012] X is halo, H₂O, (R′) (R″) S(O), R′CO₂ or (R′) (R″)C═O, where R″ represents alkyl, aryl or alkaryl and R′ is as defined above;

[0013] Y is a counterion;

[0014] m is 0 or 1;

[0015] q is 1, 2 or 3;

[0016] C′ is C₁ to C₁₂ alkylene, optionally substituted in or on the alkylene chain, bound to two A groups;

[0017] p is 0 or 1 and r is 1 when p is 0 and r is 2 when p is 1; and

[0018] A and B are each independently O-donor, N-donor or S-donor ligands and one of A and B may be halo.

[0019] Suitably, A and B are each independently N-donor nitrile ligands; or B is halo and A is an N-donor pyridine ligand, optionally substituted at one or more of the carbon rings of the pyridine ring; or B is halo and A is an 0-donor carboxylate ligand; or B is halo and A is an S-donor sulfonyl ligand; or p is 0, A is NR⁷R⁸ and B is NR⁹R¹⁰, wherein R⁷, R⁸, R⁹ and R¹⁰ independently represent H or alkyl, and A and B are linked by an alkylene chain, optionally substituted in or on the alkylene chain; or p is 1, A is NR⁷ and B is NR⁹R¹⁰, wherein R⁷, R⁹ and R¹⁰ are as previously defined, and A and B are linked by an alkylene chain, optionally substituted.

[0020] The compounds of the invention may be in the form of solvates and/or prodrugs. Prodrugs are variants of the compounds of the invention which can be converted to compounds of formula (I) in vivo.

[0021] The compounds of formula (I) may have one or more chiral centres. When the compounds of formula (I) have one or more chiral centres, they may be in the form of one enantiomer, may be enriched in one enantiomer or may be a racemic mixture.

[0022] The term “alkyl” as used herein includes C₁ to C₆ alkyl groups which may be branched or unbranched and may be open chain or, when they are C₃ to C₆ groups, cyclic. Unbranched open chain alkyl groups include, for example, methyl, ethyl, propyl, butyl, pentyl and hexyl. Branched open chain alkyl groups include, for example, 2-propyl, 2-butyl and 2-(2-methyl)propyl. Cyclic groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. The alkyl groups in the compounds of the invention may optionally be substituted. Substituents include one or more further alkyl groups and/or one or more further substituents, such as, for example, cyano, nitro, hydroxyl, haloalkyl, —CO₂alkyl, halo, thiol (SH), thioether (eg, S-alkyl) and sulfonate. The term “alkylene” is defined similarly to the definition of the term “alkyl” but includes C₂ to C₁₂ groups and is a divalent species with radicals separated by two or more (eg, from two to twelve) carbon atoms linked in a chain. Preferably, the alkylene groups are straight chain groups. Alkylene groups are optionally substituted in the akylene chain, preferably with one or more phenylene (eg, 1-4-phenylene) and/or —CONR^(1a)- groups and/or —NR^(2a)- groups, where R^(1a)and R^(2a) independently represent H, alkyl, aryl or alkaryl. Preferably, R^(1a) and R^(2a) are H or C₁ to C₃ alkyl.

[0023] The term “aryl” as used herein includes aromatic carbocyclic rings such as phenyl and naphthyl and heterocyclic rings such as pyridyl, imidazolyl, pyrrolyl and furanyl. Aryl groups may optionally be substituted with one or more substituents including, for example, alkyl, cyano, nitro, hydroxyl, haloalkyl, —CO₂alkyl, halo, thiol (SH), thioether (eg, S-alkyl) and sulfonate.

[0024] The term “alkaryl” means alkyl substituted with aryl eg, benzyl.

[0025] The term “alkether” means alkyl substituted with either —O—or —S—(eg, O-alkyl).

[0026] The term “halo” means a halogen radical selected from fluoro, chloro, bromo and iodo.

[0027] The term “haloalkyl” means alkyl substituted with one or more halo groups eg, trifluoromethyl.

[0028] The term “carboxyester” means —CO₂alkyl, —CO₂ aryl, —OCOalkyl or —OCOaryl, preferably —CO₂ alkyl or —OCOalkyl.

[0029] The term “heterocyclic ring” as used herein refers to a 3-, 4-, 5-, 6-, -7, or 8- (preferably 5-, 6- or 7-) membered saturated or unsaturated ring, which may be aromatic or non-aromatic, containing from one to three heteroatoms independently selected from N,O and S, eg, indole.

[0030] The term “carbocyclic ring” as used herein refers to a saturated or unsaturated ring, which may be aromatic or non-aromatic, containing from 3 to 8 carbon atoms (preferably 5 to 7 carbon atoms) and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

[0031] In one aspect, R¹ and R² together with the ring to which they are bound in compounds of formula (1) may represent an ortho- or peri-fused carbocyclic or heterocyclic ring system.

[0032] R¹ and R² together with the ring to which they are bound may represent a wholly carbocyclic fused ring system such as a ring system containing 2 or 3 fused carbocyclic rings eg, optionally substituted, optionally hydrogenated naphthalene or anthracene.

[0033] In another aspect, R¹ and R² together with the ring to which they are bound in compounds of formula (I) may represent a fused tricyclic ring such as anthracene or a mono, di, tri, tetra or higher hydrogenated derivative of anthracene. For example, R¹ and R² together with the ring to which they are bound in formula (I) may represent anthracene, 1, 4 dihydroanthracene or 1, 4, 9, 10-tetrahydroanthracene.

[0034] In a further aspect, R¹ and R² together with the ring to which they are bound in formula (I) may represent:

[0035] In the compounds of formula (I), R³, R⁴, R⁵ and R⁶ may represent H.

[0036] In one aspect, A and B in the compounds of formula. (I) both represent R¹¹—CN. R¹¹ is alky, preferably C₁ to C₃ alkyl, more preferably methyl.

[0037] In another aspect, one of A and B in the compounds of formula (I) represents a R¹²R¹³S(O) group and the other represents halo, preferably chloro. R¹² and R¹³ are alkyl, preferably methyl.

[0038] In a further aspect, A and B may together represent NR⁷R⁸—(CR¹⁴R¹⁵)_(n)—NR⁹R¹⁰, wherein R¹⁴ and R¹⁵ are independently H or alkyl or R¹⁴ and R¹⁵ groups, on the same carbon atom or on neighboring carbon atoms, are linked to form a carbocylic ring and n is an integer from 1 to 4. Preferably, R¹⁴ and R¹⁵ are both hydrogen and n is 2 or 3, more preferably 2. R⁷, R⁸, R⁹ and R¹⁰ are preferably H or methyl and, more preferably, all of R⁷, R⁸, R⁹ and R¹⁰ are H.

[0039] When R⁸ is present in A, then p is 0. When R⁸ is absent, then p is 1 and C′ takes the place of R⁸.

[0040] In a further aspect of the invention, R⁸ is absent from A, p is 1 and C′ is C₄ to C₁₀ straight chain alkylene (eg hexylene). Compounds according to this aspect of the invention are so-called dinuclear complexes comprising two ruthenium atoms per complex.

[0041] Other examples of dinuclear complexes of the invention are those in which pairs of A and B together with linker C′ represent:

[0042] wherein each n′, n″, x′, x″ and y′ independently represents an integer from 1 to 12, preferably 1 to 6.

[0043] Y^(q−)in compounds of formula (I) is a counterion and is only present in the compound when the complex containing the metal ion is charged. Y^(q−)is preferably a non-nucleophilic anion such as PF₆—, for example.

[0044] R′ and R″ are preferably alkyl. Most preferably, both R′ and R″ are methyl.

[0045] A particular sub-group of compounds of formula I, which may be active against resistant cell lines, are those in which R₃, R₄, R₅ and R₆ are all H, R¹ and R² together with the phenyl ring to which they are bound form an optionally hydrogenated anthracene ring system (such as O₁₄H₁₄ or C₁₄H₁₂), X is halo, A and B are N donor ligands, p is 0, r is 1, m is 1 and Y^(q−)is a non-nucleophilic ion such as PF₆—. Preferably, A and B are both NH2 groups linked by a C₂-C₆ alkylene chain, more preferably a C₂ alkylene chain ie, A and B together represent ethylenediamine.

[0046] Compounds of formula (I) may be used in medicine. In particular, compounds of formula (I) may be used to treat and/or prevent cancer.

[0047] Therefore, the present invention also provides the use of a compound of the invention (ie, a compound of formula (I)) in the manufacture of a medicament for the treatment and/or prevention of cancer.

[0048] Further provided by the invention is a method of treating and/or preventing cancer which comprises administering to a subject a therapeutically effective amount of a compound of the invention.

[0049] The compounds of the invention may be used directly against a tumour. Alternatively or additionally, the compounds may be used to prevent or inhibit metastasis and/or to kill secondary tumours. It will be understood that the prevention or inhibition of metastasis is encompassed by the term “preventing cancer”, as used herein.

[0050] Compounds of the invention may be effective in treating and/or preventing tumours caused by cells that are resistant to other cytotoxic drugs, such as cis-platin, for example.

[0051] The invention also provides a pharmaceutical composition comprising one or more compounds of the invention together with one or more pharmaceutically acceptable excipients. Suitable excipients include diluents and/or carriers.

[0052] The compounds of the invention may be administered by a number of routes including, for example, orally, parenterally (eg, intramuscularly, intravenously or subcutaneously), topically, nasally or via slow releasing microcarriers. Thus, suitable excipients for use in the pharmaceutical compositions of the invention include saline, sterile water, creams, ointments, solutions, gels, pastes, emulsions, lotions, oils, solid carriers and aerosols.

[0053] The compositions of the invention may be formulated in unit or sub-unit dosage form including, for example, tablets, capsules and lozenges and containers containing the composition in a form suitable for parenteral administration.

[0054] The specific dosage level of the compounds and compositions of the invention will depend upon a number of factors, including the biological activity of the specific compound used and the age, body weight and sex of the subject. It will be appreciated that the subject may be a human or a mammalian animal.

[0055] The compounds and compositions of the invention can be administered alone or in combination with other compounds. The other compounds may have a biological activity which complements the activity of the compounds of the invention eg, by enhancing its effect in killing tumours or by reducing any side-effects associated with the compounds of the invention.

[0056] The present invention also provides a process for preparing the compounds of the invention which comprises the reaction of a compound of formula [(η⁶—C₆(R¹)(R²)(R³)(R⁴)(R⁵)(R⁶))RuX₂] which may be in the form of a monomer or a dimer, with A and B, optionally in the presence of Y^(q−), in a suitable solvent for the reaction, wherein R¹, R², R³, R⁴, R⁵, R⁶, X, A, B and Y are as defined above for the compounds of the invention.

[0057] Suitable compounds of formula [(η⁶—(R¹)(R²)(R³)(R⁴)(R⁵)(R⁶))RuX₂] for use as starting materials (starting ruthenium complexes) in the process of the invention include [(η⁶—C₁₄)RuCl₂]₂, [(η⁶—C₁₄H₁₄)RuBr₂]₂, [(η⁶—C₁₄H₁₄)RuI₂]₂, [(η⁶—C₁₄H₁₂)RuCl₂]₂, [(η⁶—C₁₄H₁₂)RuBr₂]₂ and [(η⁶—C₁₄H¹²)RuI₂]₂ which may be prepared according to the procedures herein disclosed.

[0058] When A and B in the compounds of the invention are R¹¹—CN, the solvent for the reaction may be R¹¹—CN itself. Preferred reaction conditions include stirring the starting ruthenium complex, as described above, in R¹¹—CN as solvent at 60° C. filtering off the NH₄Cl precipitate formed and evaporating the filtrate to yield the product. The reaction mixture comprises a source of Y^(q−), such as a compound of formula (NH₄ ⁺)Y^(q−)eg, NH₄ PF⁶⁻.

[0059] Compounds of formula (I) in which A and B represent, together, NR⁷R⁸—(CR¹⁴R¹⁵)_(n)—NR⁹R¹⁰ or NR⁹R¹⁰—(CR¹⁴R¹⁵)_(n)—NR⁷—C′—NR⁷—(CR¹⁴R¹⁵)_(n)—NR⁹R¹⁰ can be produced, according to the process of the invention, by stirring the starting ruthenium complex in the presence of a slight excess of NR⁷R⁸ —(CR¹⁴R¹⁵)_(n)—NR⁹NR¹⁰ or a molar equivalent amount of NR⁹R¹⁰—(CR¹⁴R¹⁵)_(n)—NR⁷—C′—NR⁷—(CR¹⁴R¹⁵)_(n)—NR⁹R¹⁰, respectively, in a suitable solvent, preferably an alcoholic solvent such as methanol. The reaction may be carried out at room temperature or at elevated temperature (eg, 30° C. to 90° C.) until a sufficient amount of product is formed; optionally after cooling the reaction mixture. The reaction mixture comprises a source of Y^(q−), such as a compound of formula (NH₄ ⁺)Y^(q−)eg, NH₄ PF₆.

[0060] Compounds of formula (I) in which A or B is an N-donor pyridine ligand may be obtained, according to the process of the invention, by heating a mixture of the starting ruthenium complex and excess pyridine compound (such as a 1.5- to 3- fold molar excess) in a suitable solvent such as benzene until a sufficient amount of product is formed. The reaction may be carried out under reflux conditions.

[0061] Compounds of formula (I) in which A or B is an S-donor sulfonyl ligand may be obtained, according to the process of the present invention, by dissolving the starting ruthenium complex in a solution of the sulfonyl compound, eg, dimethyl sulfoxide, and diffusing the resulting coloured solution with a suitable solvent, eg, diethyl ether.

[0062] The precipitate which is formed in the process of the invention comprises or consists of the compound of the invention, The compound of the invention may be isolated from the reaction mixture by separating the precipitate from the liquid phase (eg, by filtration) and then removing the solvent from the precipitate (eg, under reduced pressure). The solid thus formed, which comprises or consists of the compound of the invention may, optionally, be purified eg, by recrystallisation from a suitable solvent (including, for certain compounds of the invention, acetonitrile or acetonitrile/ether (where A and B are R¹¹—CN and R¹¹ is methyl) and methanol/ether).

[0063] The following non-limiting examples illustrate the present invention.

EXAMPLES

[0064] A. Synthesis

[0065] General

[0066] Ethylenediamine was freshly distilled over Na, ethanol and methanol dried over P₂O₅. Tetrahydrofuran (F) was dried by distillation from Na-benzophenone.

[0067] Starting Materials

[0068] Preparation of 1,4,5,8,9,10-Hexahydroanthracene (C₁₄H₁₆)¹

[0069] Anthracene (4.0 g, 22.4 mmol) was dissolved in freshly dried THF (200 ml) and ethanol (40 ml). This mixture was added to liquid NH₃ (500 ml) which had been condensed under argon into a 1 litre flask equipped with a Dewar condenser, cooling bath (dry-ice/acetone) and mechanical stirrer. Sodium (10.40 g, 0.45 mol) was added in small pieces over a period of 20 min. After a further 50 min stirring at −60° C., the cooling bath was removed and the ammonia was allowed to evaporate under an argon flow with stirring. Into the residue was added 50 ml water slowly to decompose the excess of sodium and then a further 150 ml. This was extracted with diethyl ether (4×250 ml) and the combined ether layers washed with saturated NaCl solution (2×250 ml) and dried over MgSO₄. Removal of diethyl ether on the rotary evaporator afforded the white solid. Recrystallised (2×) first from benzene-chloroform (1:1) and then from benzene only to yield a white needle product, 98% pure by ¹H NMR. This was used without further purification.

[0070] Yield: 1.54 g, 8.36 mmol, 37.3%

[0071] Preparation of 1,4,9,10-Tetrahydroanthracene (C₁₄H₁₄)²

[0072] 9,10-Dihydroanthracene (5.0 g, 27.74 mmol) dissolved in 300 ml THF was added to refluxing ammonia which had been condensed under argon into a 1 litre flask equipped with a Dewar condenser, cooling bath (dry-ice/acetone) and mechanical stirrer. Li wire (0.48 g, 69.35 mmol) was added in small pieces over a period of 20 min. After refluxing for 4 h with stirring, to the reaction mixture was added 60 ml ethanol and then 120 ml water and the ammonia allowed to evaporate. This was extracted with diethyl ether (2×250 ml) and the combined ether layers washed with saturated NaCl solution (1×250 ml) and dried over MgSO₄. Removal of ether on the rotary evaporator afforded a light yellow solid which was recrystallized (2×) from benzene to remove most of the hexahydroanthracene (C₁₄H₁₆) as white needles. Further recrystalliation from acetone yielded white plates of the tetrahydroanthracene (C₁₄H₁₄), 97% pure by ¹H NMR. This was used without further purification.

[0073] Yield: 1.5 g, 8.23 mmol, 29.7%

[0074] Preparation of [(η⁶—C₁₄H₁₄)RuCl₂]₂ ³

[0075] 1,4,5,8,9,10-Hexahydroanthracene (1.0 g, 5.43 mmol) was added to a filtered solution of RuCl₃3H₂O (0.84 g, 3.18 mmol) in dry ethanol (60 ml). The reaction was heated to reflux under argon for 48 hours. Filtration of the warm reaction mixture left a yellow-brown solid which was washed with a little ethanol, followed by diethyl ether (4×10 ml) and dried in vacuo.

[0076] Yield: 0.96 g, 1.36 mmol, 8.5%

[0077] Preparation of [(η⁶—C₁₄H₁₂)RuCl₂]₂

[0078] 1,4,9,10-Tetrahydroanthracene (0.45 g, 2.49 mmol) was added to a filtered solution of RuCl₃3H₂O (0.48 g, 1.83 mmol) in dry ethanol (45 ml). The reaction was heated to reflux under argon for 48 h. Filtration of the warm reaction mixture left a brown solid which was washed with a little ethanol, followed by diethyl ether (4×10 ml) and dried in vacuo.

[0079] Yield: 0.57 g, 0.81 mmol, 88.5%

Example 1

[0080] Preparation of [(η⁶—C₁₄H₁₄)RuCl(en)]⁺PF₆

[0081] [(η⁶—C₁₄H₁₄)RuCl₂]₂(0.205 g, 0.289 mmol) was stirred in dry methanol (25 ml) under argon at 60° C. Ethylenediamine (en) (48 μl, 0.75 mmol) was added in one portion. The reaction was stirred at 60° C. for 3 h and filtered and NH₄PF₆ (0.4 g, 2.45 mmol) added. The volume was reduced to approximately 6 ml on the rotary evaporator. After standing at 4° C. overnight, the yellow microcrystalline solid was collected, washed with a little methanol, followed by ether and dried in vacuo. This was recrystallised from methanol/ether.

[0082] Yield: 0.1 g, 0.19 mmol, 32.9%

[0083] C₁₆H₂₂CIF₆N₂PRu(523.85) Calc. % C=36.68 % H=4.23 % N-5.35 Found % C=36.20 % H=4.17 % N=5.34

Example 2

[0084] Preparation of [(η⁶—C₁₄H₁₄)RuCl₂(DMSO)]³

[0085] [(η⁶—C₁₄H₁₄)RuCl₂]₂ (0.05 g, 0.07 mmol) was dissolved into dimethyl sulfoxide (2 ml) and filtered to yield a deep red solution. Slow diffusion of diethyl ether into this solution resulted in the formation of brilliant red crystals suitable for X-ray diffraction. The crystals were collected and washed thoroughly with diethyl ether (4×10 ml).

[0086] Yield: 0.03 g, 0.07 mmol, 49.5%

[0087] C₁₆H₂₀Cl₂ORuS(432.37) Calc. % C=44.45 % H=4.66 Found % C=44.41 % H=4.51

Example 3

[0088] Preparation of [(η⁶—C₁₄H₁₄)RuCl(CH₃CN)₂]⁺PF₆—

[0089] [(η⁶—C₁₄H₁₄)RuCl₂]₂ (0.10 g, 0.144 mmol) was suspended in 10 ml acetonitrile. NH₄PF₆ (47.1 mg, 0.288 mmol) in 2 ml acetonitrile was added in one portion. The reaction was stirred at 60° C. without special precautions to exclude air. After 48 h the pale brown precipitate was filtered off and orange filtrate evaporated to yield an orange solid. This was recrystallized from acetonitrile/ether to yield orange crystals.

[0090] Yield: 0.13 g, 0.238 mmol, 82.8%

[0091] C₁₈H₂₀ClF₆N₂PRu(545.86) Calc. % C=39.60 % H=3.69 % N=5. 13 Found % C=39.17 % H=3.48 % N=5.47

Example 4

[0092] Preparation of [η⁶—C₁₄H₁₂)RuCl(en)]⁺PF₆—

[0093] [(η⁶—C₁₄H₁₂)RuCl₂]₂ (0.10 g, 0.142 MMol) was stirred in 10 ml dry methanol under argon at 60° C. Ethylenediamine (en) (24 μl, 0359 mmol) was added in one portion. The reaction was maintained at 60° C. with stirring for 5 h and filtered. The volume was reduced to approximately 4 ml on the rotary evaporator and then a solution of NH₄PF₆ (0.20 g, 1.227 mmol) in 2 ml methanol was added. A yellow solid precipitated from the mixed solution when briefly shaken. After standing at 4° C. overnight, this solid was collected, washed with a little methanol, followed by diethyl ether and dried in vacuo. This was recrystallised from benzylalcohol/ether.

[0094] Yield: 0.1 g, 0.19 mmol, 67.5%

[0095] C₁₆H₂₀ClF₆N₂PRu(521.83) Calc. % C=36.82 % H=3.86 % N=5.37 Found % C=36.50 % H=3.85 % N=5.38

Example 5

[0096] Preparation of [(η⁶—C₁₄H₁₂)RuCl₂(DMSO)]

[0097] [(η⁶—C₁₄H₁₂)RuCl₂]₂ (0.05 g, 0.07 mmol) was dissolved into dimethyl sulfoxide (1 ml) and filtered to yield a rose red solution. Slow diffusion of diethyl ether into this solution resulted in the formation of brilliant red crystals suitable for X-ray diffraction. The crystals were collected and washed thoroughly with diethyl ether (3×10 ml).

[0098] Yield: 0.025 g, 0.058 mmol, 41.4%

[0099] C₁₆, H₁₈Cl₂ORuS(430.35) Calc. % C=44.65 % H=4.21 Found % C=44.08 % H=4.18

Example 6

[0100] Preparation of [(η⁶—C₁₄H₁₂)RuCl(CH₃CN)₂]⁺PF₆—

[0101] [(η⁶—C₁₄H₁₂)RuCl₂]₂ (0.10 g, 0.142 mmol) was suspended in 10 ml acetonitrile. NH₄PF₆ (48.6 mg, 0.298 mmol) in 2 ml acetonitrile was added in one portion. The reaction was stirred at 60° C. without special precautions to exclude air. After 25 h the pale brown precipitate was filtered off and the orange filtrate evaporated to yield an orange solid. This was recrystallized from acetonitrile/ether to yield orange crystals.

[0102] Yield: 0.125 g, 0.23 mmol, 81%

[0103] C₁₈H₁₈ClF₆N₂PRu(543.85) Calc. % C=39.75 % H=3.34 % N=5.15 Found % C=39.42 % H=3.33 % N=5.14

Example 7

[0104] Preparation of {[(η⁶—C₁₄H₁₄)RuCl]₂[H₂N(CH₂)₂NH(CH₂)₆NH(CH₂)₂NH₂—N,N′N″,N″′]}²⁺.2PF₆—

[0105] The starting material [(η⁶—C₁₄H₁₄)RuCl₂]₂ was prepared as previously described. Ethylenediamine and triethylamine were freshly distilled over Na. Tetrahydrofuran (THP) was dried by distillation from Na-benzophenone. Triphenylmethyl chloride (99%) and adipoyl chloride (98%) were purchased from the Arcos Chemical Co. All other chemicals were AR grade and were used as received.

[0106] (a) N-tritylethyldiamine

[0107] A solution of trityl chloride (5.57 g, 20 mmol) in dichloromethane (25 ml) was slowly added into a solution of ethylenediamine (8 ml, 120 mmol) in dichloromethane (75 ml) with stirring at room temperature. The addition was accomplished within 1 h and the reaction stirred overnight. The white salt was filtered off and the filtrate washed with water and dried over anhydrous sodium sulphate. Dichloromethane was removed by rotary evaporation and the residue dissolved into methanol. A white precipitate began to form after shaking for a while and the mixture was kept in the refrigerator for 5 h and then filtered off. The methanol filtrate was reduced to 10 ml and kept in the refrigerator overnight. A white solid precipitated. This was collected as the desired product and washed with diethyl ether and dried in vacuo.

[0108] Yield: 4.5 g, 14.88 mmol, 74.4%

[0109] (b) N,N′-Bis(2′-tritylaminoethyl)-1,6-diamidohexane

[0110] N-Tritylethyldiarine (1.5 g, 4.96 mmol) and triethylamine (1.0 g, 7.29 mmol) were dissolved in chloroform (35 ml) and cooled in an ice bath. To this solution was added adipoyl chloride (0.36 ml, 2.48 mmol) in chloroform (10 ml) slowly with stirring. After addition, the mixture was refluxed for 2 h and cooled to room temperature. This was filtered to give a clear chloroform filtrate (see below). The filtered precipitate was dissolved into dichloromethane. This was washed with water and then saturated NaCl solution and dried over anhydrous sodium sulphate. Removal of the solvent by rotary evaporation gave a white product. The chloroform filtrate was also washed with water and saturated NaCl solution and dried over anhydrous sodium sulphate. After removal of chloroform, a further crop of product was obtained.

[0111] Yield: 1.40 g, 1.91 mmol, 77%

[0112] C₄₈,H₅O₂N₄H₂O(732.96) Calc. % C=78.66 % H=7.15 % N=7.64 Found % C=78.81 % H=6.73 % N=7.55

[0113] (c) N,N′-Bis(2′-tritylaminoethyl)-1,6-diaminohexainie

[0114] To a solution of N,N′-bis(2′-tritylaminoethyl)-1,6-diamidohexane (1.3 g, 1.82 mmol) in dry THF was added a suspension of L (0.69 g, 18.18 mmol) in dry THF (20 ml) under argon with vigorous stirring. After the addition, the reaction was heated to a gentle reflux with stirring for 25 h. This was cooled to 4° C. The reaction product and excess hydride were decomposed by the dropwise addition of H₂O (0.69 ml), followed by 15% (w/v) NaOH solution (0.69 ml) and H₂O (2.07 ml) in succession. After vigorous stirring for 30 min, the mixture was filtered by suction and the resulting cake was washed thoroughly with dichloromethane. The combined filtrate was concentrated to dryness on the rotary evaporator and the resulting residue dissolved into dichloromethane (50 ml). This was washed with water and then saturated NaCl solution and dried over anhydrous sodium sulphate. Removal of dichloromethane by rotary evaporator afforded a colourless solid.

[0115] Yield: 1.20 g, 1.75 mmol, 96%

[0116] (d) N,N′-Bis(2-aminoethyl),6diaminohexane tetrahydrochloride

[0117] A mixture of N, N′-bis(2′-tritylaminoethyl)1,6-diaminohexane (1.0 g 1.45 mmol) and 6 M HCl (30 ml) was refluxed for 3 h. The mixture was filtered and the filtrate was concentrated to about 3 ml over vacuao. Addition of methanol into the concentrated solution afforded a white salt.

[0118] Yield: 0.46 g, 1.32 mmol, 92%

[0119] C₁₀H₂₆N₄.4HCl(348.09) Calc. % C=34.48 % H=8.68 % N=16.09 Found % C=34.26 % H=8.77 % N=16.24

[0120] (e) {[(η⁶—C₁₄H₁₄)RuCl]₂ [H₂N(CH₂)₂NH(CH₂)₆NH(CH₂)₂—N,N′,N″,N″′]}²⁺.2PF₆—

[0121] [(η⁶—C₁₄H₁₄)RuCl₂]₂ (0.15 g, 0.213 mmol) in 10 ml methanol was stirred under argon at 60° C. To this suspension was added a solution of N,N′-bis(2-aminoethyl)-1,6-diaminehexane (0.213 mmol) in methanol which was obtained by treatment of N,N′-bis(2-aminoethyl)1,6-diaminehexane tetrahydrochloride (73.97 g, 0.213 mmol) with 1.697 ml 0.5008 N KOH—MeOH solution. The mixture was stirred at 60° C. for a further 1.5 h. This was filtered while hot and concentrated to 6 ml. Addition of NH₄PF₆ (0.25 g; 1.53 mmol) to the concentrated solution afforded a yellow precipitate. This was recrystallized from methanol/ether.

[0122] Yield: 0.09 g, 0.0796 mmol, 37.4%

[0123] C₃₈H₅₄Cl₂F₁₂N₄P₂RU₂(1129.85) Calc. % C=40.39 % H=4.81 % N=4.95 Found % C=40.30 % H=4.49 % N=4.21

[0124] B. Biological Data

[0125] 1. Protocol for Testing Ru Compounds

[0126] The compounds are tested on 24 well trays. Cells growing in a flask are harvested just before they become confluent, counted using a haemocytometer and diluted down with media to a concentration of 1×10⁴ cells per ml. The cells are then seeded in the 24 well trays at a density of 10×10 ⁴ cells per well (i.e. 1 ml of the diluted cell suspension is added to each well). The cells are then left to plate down and grow for 72 hours before adding the compounds of the invention.

[0127] The Ru complexes are weighed out and made up to a concentration of is 1 mg/ml with deionised water then sonicated, until they go into solution. The appropriate volume of the Ru solution is added to 5 ml of media to make it up to a concentration of 100 μM for each drug. This 100 μM solution is then serially diluted to make up the 10 μM, 1 μM and 0.1 μM solutions.

[0128] The media is removed from the cells and replaced with 1 ml of the media dosed with drug. Each concentration is done in duplicate. A set of control wells are left on each plate, containing media without drug.

[0129] The cells are left exposed to the drugs for 24 hours and then washed with phosphate buffered saline before fresh media is added.

[0130] They are allowed to grow on for a further 3 days before being counted using a Coulter counter.

[0131] Preparing cells for counting: Media is removed and 1 ml of PBS is added to the cells. 250 μl of trypsin is added and cells left in incubator for a few minutes to allow the monolayers to detach. Once trypsinised, 250 μl of media is added to each well to neutralise the trypsin. 200 μl of this suspension is added to 10 ml of NaCl for counting.

[0132] 2. Results

[0133] Using the above protocol, a number of compounds of the invention were tested on A2780 ovarian cancer cell line. The results are as follows: Compound (Example No.) IC50 (μM) 1 0.5 2 94 3 177 4 0.3 5 68 6 315 7 6

[0134] The experiments were repeated to investigate the effect of the compounds of the invention on drag-resistant variants of the A2780 cell line. The following results were obtained: Compound IC50 (μM) (Example No.) A2780 A2780 cis* A2780 AD** 1 0.5 1 328 2 94 493 4 3 116 2 2 4 2 16 104 5 126 2 5 6 192 2 0.9

[0135] Compounds of the invention therefore have cytotoxicity against cancer cells that are resistant to treatment by other drugs.

REFERENCES

[0136]¹A. J. Birch, P. Fitton, D. C. C. Smith, D. E. Steere, A. R. Stelfox J. Chem. Soc. 1963, 2209-2216

[0137]² R. G. Harvey J. Org. Chem. 1967, 32, 238

[0138]³ T. J. Beasley, R. D. Brost, C. K. Chu, S. L. Grundy, S. R. Stobart Organometallics 1993, 12, 4599-4606 

1. Ruthenium (II) compound of formula (I):

wherein R¹ and R² together with the ring to which they are bound represent a saturated or unsaturated carbocyclic or heterocyclic group containing up to three 3- to 8- membered carbocyclic or heterocyclic rings, wherein each carbocyclic or heterocyclic ring may be fused to one or more other carbocyclic or heterocyclic rings; and wherein each of the rings may be optionally substituted by one or more groups independently selected from alkyl, aryl, alkaryl, halo, carboxy, carboxyester, carboxyamide, sulfonate, sulfonamido or alkether; R³, R⁴, R⁵ and R⁶ independently represent H, alkyl, —CO₂R′, aryl or alkaryl, which latter two groups are optionally substituted on the aromatic ring; R′ represents alkyl, aryl or alkaryl; X is halo, H₂O, (R′) (R″) S(O), R′ CO₂ ⁻or (R′) (R″)C═O, where R″ represents alkyl, aryl or alkaryl; Y is a counterion; m is 0 or 1; q is 1, 2 or 3; C′ is C₁ to C₁₂ alkylene, optionally substituted in or on the alkylene chain, bound to two A groups; p is 0 or 1 and r is 1 when p is 0 and r is 2 when p is 1; and A and B are each independently O-donor, N-donor or S-donor ligands and one of A and B may be halo.
 2. Compound as claimed in claim 1, wherein R³, R⁴, R⁵ and R⁶ all represent H.
 3. Compound as claimed in claim 1 or claim 2, wherein R¹ and R² together with the ring to which they are bound represent anthracene.
 4. Compound as claimed in claim 3, wherein R¹ and R² together with the ring to which they are bound represent 1,4-dihydroanthracene.
 5. Compound as claimed in claim 3, wherein R¹ and R² together with the ring to which they are bound represent 1,4,9,10-tetrahydroanthracene.
 6. Compound as claimed in any one of claims 1 to 5, wherein A and B are both R¹¹—CN and R¹¹ represents alkyl.
 7. Compound as claimed in any one of claims 1 to 5, wherein one of A and B is a R¹²R¹³S(o) group and the other is halo.
 8. Compound as claimed in any one of claims 1 to 5, wherein A and B together represent NR⁷R⁸—(CR¹⁴R¹⁵)_(n)—NR⁹R¹⁰, wherein R¹⁴ and R¹⁵ are hydrogen, or are linked at the same or neighbouring carbon atoms to form a carbocylic ring, and n is an integer from 1 to
 4. 9. Compound as claimed in claim 8, wherein R⁷, R⁸, R⁹ and R¹⁰ all represent H.
 10. Compound as claimed in claim 8 or claim 9, wherein R¹⁴ and R¹⁵ are both H and n is
 2. 11. Compound as claimed in any one of claims 8 to 10, wherein p is
 0. 12. Compound as claimed in any one of claims 8 to 10, wherein R⁸ is absent, p is 1 and C′ is C₄ to C₁₀ straight chain alkylene.
 13. Compound of any one of claims 1 to 12 for use in medicine.
 14. Use of a compound of any one of claims 1 to 12 in the manufacture of a medicament for the treatment and/or prevention of cancer.
 15. Pharmaceutical composition comprising a compound of any one of claims 1 to 12 together with one or more pharmaceutically acceptable excipients.
 16. A method of treating and/or preventing cancer which comprises administering to a subject a therapeutically effective amount of a compound of any one of claims 1 to 12 or a composition of claim
 15. 17. Process for preparing the compound of any one of claims 1 to 12 which comprises the reaction of a compound of formula [(η⁶—C₆(R¹)(R²)(R³)(R⁴)(R⁵)(R⁶))RuX₂], optionally in the form of a dimer, with A and B, optionally in the presence of Y^(q−), in a suitable solvent for the reaction, wherein R¹, R², R³, R⁴, R⁵, R⁶, X, A, B, q and Y are as defined in Claim
 1. 